Chronic hepatitis B (CHB) affects nearly 400 million people worldwide, with ~1 million annual deaths due to liver disease and hepatocellular carcinoma. HIV-1/hepatitis B virus (HBV) co-infection is common. HIV-1 increases liver disease progression from CHB with liver disease being a leading cause of mortality in HIV-1 infected persons taking antiretroviral therapy (ART). Thus, a cure for CHB is needed. Although dually active ART (DAART) controls HBV and HIV-1 viremia, it cannot cure CHB because it does not eradicate the stable covalently closed circular DNA (cccDNA) form of HBV, the template for replication, from the hepatocyte. Our preliminary data demonstrate that some hepatocytes contain cccDNA but have limited viral transcription (no pregenomic RNA (pgRNA)), which we define as transcriptionally `silent' HBV-infected hepatocytes (tsHBiH). These cells may be functionally `cured' temporarily, but may also be the cells that reactivate. Understanding mechanisms of transcriptional silencing can elucidate intracellular processes that can be exploited to convert transcriptionally active HBV-infected hepatocytes (taHBiH) to tsHBiH and potentially lead to a functional cure. We propose comparing tsHBiH and taHBiH by dissecting single hepatocytes with single-cell laser capture microdissection (scLCM) from an established cohort of 21 HIV-HBV co-infected people who have long- standing control of HBV with DAART with archived liver tissues at two time points during DAART. In aim 1, scLCM and digital droplet PCR (ddPCR) will be used to quantify HBV replicative forms in thousands of individual hepatocytes from our cohort to determine the proportion that are tsHBiH or taHBiH. This aim further tests how HIV-1 associated immune dysregulation affects the burden of these cells. In aim 2, we test if intracellular innate immune molecules maintain tsHBiH by quantifying intracellular innate immune mRNAs. We also test alternative mechanisms such as defective virus or epigenetic silencing of cccDNA, or other viral factors. In aim 3, we use the paired liver biopsies to compare the decline rates of tsHBiH and taHBiH and test whether tsHBiH persist longer. We also determine if HIV-1 related immune dysregulation affects decline rates. Innovative aspects of this proposal include scLCM and ddPCR. Our research will provide the first quantitations of cccDNA and pgRNA in single hepatocytes in humans, and reveal mechanisms underlying HBV transcription. Relevance HBV is the leading cause of liver disease worldwide and is an important co-infection in HIV-1 infected individuals. Medicines can control chronic hepatitis B but are lifelong because they do not affect the stable genetic viral material in the principal liver cell, the hepatocyte. We aim to understand hepatocytes that are transcriptionally silently-infected (infected but not replicating virus) and the mechanisms of silencing by studying HBV viral forms from thousands of hepatocytes from HIV-HBV co-infected people. We will also determine how HIV-1 immune dysregulation affects the proportion of silently-infected hepatocytes.